Antifungal compounds and processes for making

ABSTRACT

The present invention relates to polymorphic forms of compound 1 or 1a and processes for preparing compound 1 and 1a polymorphs, which are useful as antifungal agents. In particular, the invention seeks to provide a new methodology for preparing polymorphs of compound 1 and substituted derivatives thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/US2016/022858, filed Mar. 17, 2016,which claims the benefit and priority of U.S. Provisional ApplicationNo. 62/135,682, filed Mar. 19, 2015, the entire contents of theaforementioned applications are hereby incorporated herein by reference.

The present invention relates to polymorphic forms of compound 1 or 1aand processes for preparing compound 1 and 1a polymorphs, which areuseful as an antifungal agents. In particular, the invention seeks toprovide a new methodology for preparing polymorphs of compound 1 andsubstituted derivatives thereof.

BACKGROUND

It is well known that the crystalline polymorph form of a particulardrug is often an important determinant of the drug's ease ofpreparation, stability, solubility, storage stability, ease offormulation and in vivo pharmacology. Polymorphic forms occur where thesame composition of matter crystallizes in a different latticearrangement resulting in different thermodynamic properties andstabilities specific to the particular polymorph form. In cases wheretwo or more polymorph substances can be produced, it is desirable tohave a method to prepare each of the polymorphs in pure form. Indeciding which polymorph is preferable, the numerous properties of thepolymorphs must be compared and the preferred polymorph chosen based onthe many physical property variables. It is entirely possible forexample that one polymorph form can be preferable in some circumstanceswhere certain aspects such as ease of preparation, stability, etc. aredeemed to be especially important. In other situations, for example, adifferent polymorph may be preferred for greater solubility and/orsuperior pharmacokinetics.

Because improved drug formulations, showing, for example, betterbioavailability or better stability are consistently sought, there is anongoing need for new or purer polymorphic forms of existing drugmolecules. The various crystalline polymorphs of Compounds 1 or 1adescribed herein help meet these and other needs.

BRIEF SUMMARY OF THE INVENTION

The invention is directed toward polymorph forms of compounds 1 or 1a.The invention is also directed toward methods of synthesis of 1 or 1a.The methods can comprise the compounds herein.

One aspect of the invention relates to polymorph forms of compounds offormula 1 or 1a, or a pharmaceutically acceptable salt, hydrate,solvate, complex or prodrug thereof.

In another aspect, the invention is an anhydrous form of a compound ofany of the formulae herein. In other aspects, the anhydrous form of acompound of any of the formulae herein is isolated. In other aspects,the anhydrous form of a compound of any of the formulae herein isisolated and has less than 2 weight % water therein (e.g., <1 wt. %,<0.5 wt. %, <0.25 wt. %, <0.1 wt. %). In other aspects, the anhydrousform of a compound of any of the formulae herein is isolated and hasless than 750 ppm water therein (e.g., <500 ppm, <400 ppm, <300 ppm,<250 ppm, <200 ppm, <150 ppm, <100 ppm, <50 ppm).

In another aspect, the invention is a salt, hydrate, or solvate form ofa compound of any of the formulae herein, having the physicochemicalcharacteristics described herein.

The compounds herein include those wherein the compound is identified asattaining affinity, at least in part, for a metalloenzyme by formationof one or more of the following types of chemical interactions or bondsto a metal: sigma bonds, covalent bonds, coordinate-covalent bonds,ionic bonds, pi bonds, delta bonds, or backbonding interactions.

Compounds 1 and 1a are described in the art, including in U.S. Pat. No.8,236,962 (incorporated by reference herein), as are methods of makingthem.

Methods for assessing metal-ligand binding interactions are known in theart as exemplified in references including, for example, “Principles ofBioinorganic Chemistry” by Lippard and Berg, University Science Books,(1994); “Mechanisms of Inorganic Reactions” by Basolo and Pearson JohnWiley & Sons Inc; 2nd edition (September 1967); “Biological InorganicChemistry” by Ivano Bertini, Harry Gray, Ed Stiefel, Joan Valentine,University Science Books (2007); Xue et al. “Nature Chemical Biology”,vol. 4, no. 2, 107-109 (2008).

In the following aspects, reference is made to the schemes and compoundsherein, including the reagents and reaction conditions delineatedherein. Other aspects include any of the compounds, reagents,transformations or methods thereof delineated in the examples herein (inwhole or in part), including as embodiments with single elements (e.g.,compounds or transformations) or embodiments including multiple elements(e.g., compounds or transformations).

In another aspect, the invention provides a method to prepare ananhydrous form of any compound of a formulae herein comprising placing acompound of a formulae herein in a solvent and recrystallizing andisolating the compound as an anhydrous form of the compound. In anotheraspect, the method further comprises: drying the resulting anhydrouscompound; drying the resulting anhydrous compound under vacuum; dryingthe resulting anhydrous compound under vacuum at about 35° C. or higher(e.g., 40° C. or higher; 45° C. or higher; 50° C. or higher).

In aspects, the method above comprises: an organic solvent (e.g.,ethers, ketones, esters, alcohols, amides, acetonitrile, and the like),an alcohol solvent (e.g., methanol, ethanol, 1-propanol, 2-propanol,butanol, and the like); two or more solvents (e.g., a combination ofalcohols, a combination of an alcohol and a hydrocarbon, 2-propanol andn-hexane); or three or more solvents (e.g., a combination of threealcohols, a combination of two alcohols and a hydrocarbon). Solventsuseful herein are known in the art.

In aspects, the method above comprises: an organic solvent (e.g.,hydrocarbons, ethers, ketones, esters, amides, acetonitrile, and thelike), an alcohol solvent (e.g., methanol, ethanol, 1-propanol,2-propanol, butanol, and the like), or water; two or more solvents(e.g., a combination of an organic solvent and an alcohol; a combinationof an alcohol and a hydrocarbon; 2-propanol and n-heptane); or three ormore solvents. In another aspect, the method comprises a combination ofsolvents, wherein said combination contains <15% (w/w) total amount ofwater, methanol, and ethanol. In another aspect, the combination ofsolvents comprises an organic solvent (e.g., hexanes, heptanes,tert-butyl methyl ether, cyclohexane, toluene, anisole, xylene,cyclohexanone, methyl-tetrahydrofuran, dimethylformamide,N-methylpyrrolidinone) and an alcohol (e.g., methanol, ethanol,1-propanol, 2-propanol, butanol), wherein said combination contains <X %(w/w) total amount of water, methanol, and ethanol. In another aspect,the combination of solvents is 2-propanol and an organic solvent. Inanother aspect, the combination of solvents is an alcohol and n-heptane,wherein said combination contains <X % (w/w) total amount of water,methanol, and ethanol. In another aspect, the combination of solvents is2-propanol and n-heptane. Solvents useful herein are known in the art.

In another aspect, the invention is an anhydrous form of a compound ofany of the formulae herein, made by a process described herein.

Another aspect of the invention relates to a process for preparing apolymorph form of compound of formula 1 or 1a, or a pharmaceuticallyacceptable salt, hydrate, solvate, complex or prodrug thereof.

In another aspect, the crystallization solvent or crystallizationsolvent mixture from any of the embodiments presented herein is ethylacetate, isopropyl acetate, ethanol, methanol, or acetonitrile, orcombinations thereof.

In another aspect, the crystallization co-solvent or crystallizationco-solvent mixture from any of the embodiments presented herein ispentane, methyl t-butylether, hexane, heptane, or toluene, orcombinations thereof.

In another aspect, any of the embodiments presented herein may compriserepeating the enantio-enrichment step(s) until desired level ofenantio-enrichment is attained.

In another aspect, any of the embodiments presented herein may compriserepeating the enantio-enrichment step(s) and/or purification steps untildesired level of enantio-enrichment and/or purification is attained.

In other aspects, the invention provides a compound of any of theformulae herein, wherein the compound inhibits (or is identified toinhibit) lanosterol demethylase (CYP51).

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of any formulae herein and a pharmaceuticallyacceptable carrier.

In other aspects, the invention provides a method of modulatingmetalloenzyme activity in a subject, comprising contacting the subjectwith a compound of any formulae herein, in an amount and underconditions sufficient to modulate metalloenzyme activity.

In one aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a metalloenzyme-related disorder ordisease, comprising administering to the subject an effective amount ofa compound or pharmaceutical composition of any formulae herein.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a metalloenzyme-related disorder ordisease, wherein the subject has been identified as in need of treatmentfor a metalloenzyme-related disorder or disease, comprisingadministering to said subject in need thereof, an effective amount of acompound or pharmaceutical composition of any formulae herein, such thatsaid subject is treated for said disorder.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a metalloenzyme-mediated disorder ordisease, wherein the subject has been identified as in need of treatmentfor a metalloenzyme-mediated disorder or disease, comprisingadministering to said subject in need thereof, an effective amount of acompound or pharmaceutical composition of any formulae herein, such thatmetalloenzyme activity in said subject is modulated (e.g., downregulated, inhibited). In another aspect, the compounds delineatedherein preferentially target cancer cells over nontransformed cells.

DESCRIPTION OF THE FIGURES

FIG. 1: depicts a XRPD pattern of an anhydrous form of compound 1.

FIG. 2: depicts a thermal analysis pattern of an anhydrous form ofcompound 1.

FIG. 3: depicts a XRPD pattern of an ethanol solvate form of compound 1.

FIG. 4: depicts a thermal analysis pattern of an ethanol solvate form ofcompound 1.

FIG. 5: depicts a XRPD pattern of a 1.5 hydrate form of compound 1.

FIG. 6: depicts a thermal analysis pattern of a 1.5 hydrate form ofcompound 1.

FIG. 7: depicts XRPD patterns of X-hydrate (upper) and anhydrous (lower)forms of compound 1.

FIG. 8: depicts thermal analysis patterns of X-hydrate (upper) andanhydrous (lower) forms of compound 1.

FIG. 9: depicts two molecules of Compound 1 in the asymmetric unit ofthe anhydrous form showing the numbering scheme employed.

FIG. 10: depicts the asymmetric unit of the ethanol solvate from thecrystal structure showing the numbering scheme employed.

FIG. 11: depicts the asymmetric unit of the 1.5 hydrate from the crystalstructure showing the numbering scheme employed.

DETAILED DESCRIPTION Definitions

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “diastereomers” refers to stereoisomers with two or morecenters of dissymmetry and whose molecules are not mirror images of oneanother.

The term “enantiomers” refers to two stereoisomers of a compound whichare non-superimposable mirror images of one another. An equimolarmixture of two enantiomers is called a “racemic mixture” or a“racemate.”

The term “isomers” or “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space.

The term “prodrug” includes compounds with moieties which can bemetabolized in vivo. Generally, the prodrugs are metabolized in vivo byesterases or by other mechanisms to active drugs. Examples of prodrugsand their uses are well known in the art (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). The prodrugs can beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form or hydroxyl with a suitable esterifying agent. Hydroxyl groupscan be converted into esters via treatment with a carboxylic acid.Examples of prodrug moieties include substituted and unsubstituted,branch or unbranched lower alkyl ester moieties, (e.g., propionoic acidesters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters(e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g.,acetyloxymethyl ester), acyloxy lower alkyl esters (e.g.,pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkylesters (e.g., benzyl ester), substituted (e.g., with methyl, halo, ormethoxy substituents) aryl and aryl-lower alkyl esters, amides,lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferredprodrug moieties are propionoic acid esters and acyl esters. Prodrugswhich are converted to active forms through other mechanisms in vivo arealso included. In aspects, the compounds of the invention are prodrugsof any of the formulae herein.

The term “subject” refers to animals such as mammals, including, but notlimited to, primates (e.g., humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice and the like. In certain embodiments, thesubject is a human.

The terms “a,” “an,” and “the” refer to “one or more” when used in thisapplication, including the claims. Thus, for example, reference to “asample” includes a plurality of samples, unless the context clearly isto the contrary (e.g., a plurality of samples), and so forth.

Throughout this specification and the claims, the words “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

As used herein, the term “about,” when referring to a value is meant toencompass variations of, in some embodiments±20%, in someembodiments±10%, in some embodiments±5%, in some embodiments±1%, in someembodiments±0.5%, and in some embodiments±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

Use of the word “inhibitor” herein is meant to mean a molecule thatexhibits activity for inhibiting a metalloenzyme. By “inhibit” herein ismeant to decrease the activity of metalloenzyme, as compared to theactivity of metalloenzyme in the absence of the inhibitor. In someembodiments, the term “inhibit” means a decrease in metalloenzymeactivity of at least about 5%, at least about 10%, at least about 20%,at least about 25%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or at least about95%. In other embodiments, inhibit means a decrease in metalloenzymeactivity of about 5% to about 25%, about 25% to about 50%, about 50% toabout 75%, or about 75% to 100%. In some embodiments, inhibit means adecrease in metalloenzyme activity of about 95% to 100%, e.g., adecrease in activity of 95%, 96%, 97%, 98%, 99%, or 100%. Such decreasescan be measured using a variety of techniques that would be recognizableby one of skill in the art. Particular assays for measuring individualactivity are described below.

Furthermore the compounds of the invention include olefins having eithergeometry: “Z” refers to what is referred to as a “cis” (same side)configuration whereas “E” refers to what is referred to as a “trans”(opposite side) configuration. With respect to the nomenclature of achiral center, the terms “d” and “1” configuration are as defined by theIUPAC Recommendations. As to the use of the terms, diastereomer,racemate, epimer and enantiomer, these will be used in their normalcontext to describe the stereochemistry of preparations.

As used herein, the term “alkyl” refers to a straight-chained orbranched hydrocarbon group containing 1 to 12 carbon atoms. The term“lower alkyl” refers to a C1-C6 alkyl chain. Examples of alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, tert-butyl, and n-pentyl.Alkyl groups may be optionally substituted with one or moresubstituents.

The term “alkenyl” refers to an unsaturated hydrocarbon chain that maybe a straight chain or branched chain, containing 2 to 12 carbon atomsand at least one carbon-carbon double bond. Alkenyl groups may beoptionally substituted with one or more substituents.

The term “alkynyl” refers to an unsaturated hydrocarbon chain that maybe a straight chain or branched chain, containing the 2 to 12 carbonatoms and at least one carbon-carbon triple bond. Alkynyl groups may beoptionally substituted with one or more substituents.

The sp² or sp carbons of an alkenyl group and an alkynyl group,respectively, may optionally be the point of attachment of the alkenylor alkynyl groups.

The term “alkoxy” refers to an —O-alkyl radical.

As used herein, the term “halogen”, “hal” or “halo” means —F, —Cl, —Bror -I.

The term “haloalkoxy” refers to an —O-alkyl radical that is substitutedby one or more halo substituents. Examples of haloalkoxy groups includetrifluoromethoxy, and 2,2,2-trifluoroethoxy.

The term “cycloalkyl” refers to a hydrocarbon 3-8 membered monocyclic or7-14 membered bicyclic ring system having at least one saturated ring orhaving at least one non-aromatic ring, wherein the non-aromatic ring mayhave some degree of unsaturation. Cycloalkyl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, or 4 atoms of each ring of a cycloalkyl group may be substituted by asubstituent. Representative examples of cycloalkyl group includecyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, cycloheptyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and thelike.

The term “aryl” refers to a hydrocarbon monocyclic, bicyclic ortricyclic aromatic ring system. Aryl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, 4, 5 or 6 atoms of each ring of an aryl group may be substituted by asubstituent. Examples of aryl groups include phenyl, naphthyl,anthracenyl, fluorenyl, indenyl, azulenyl, and the like.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, andthe remainder ring atoms being carbon (with appropriate hydrogen atomsunless otherwise indicated). Heteroaryl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, or 4 atoms of each ring of a heteroaryl group may be substituted by asubstituent. Examples of heteroaryl groups include pyridyl, furanyl,thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl thiazolyl,isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, isoquinolinyl, indazolyl, and thelike.

The term “nitrogen-containing heteroaryl” refers to a heteroaryl grouphaving 1-4 ring nitrogen heteroatoms if monocyclic, 1-6 ring nitrogenheteroatoms if bicyclic, or 1-9 ring nitrogen heteroatoms if tricyclic.

The term “heterocycloalkyl” refers to a nonaromatic 3-8 memberedmonocyclic, 7-12 membered bicyclic, or 10-14 membered tricyclic ringsystem comprising 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, S, B, P or Si, wherein the nonaromatic ring system iscompletely saturated. Heterocycloalkyl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, or 4 atoms of each ring of a heterocycloalkyl group may besubstituted by a substituent. Representative heterocycloalkyl groupsinclude piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl,thiomorpholinyl, 1,3-dioxolane, tetrahydrofuranyl, tetrahydrothienyl,thiirenyl, and the like.

The term “alkylamino” refers to an amino substituent which is furthersubstituted with one or two alkyl groups. The term “aminoalkyl” refersto an alkyl substituent which is further substituted with one or moreamino groups. The term “hydroxyalkyl” or “hydroxylalkyl” refers to analkyl substituent which is further substituted with one or more hydroxylgroups. The alkyl or aryl portion of alkylamino, aminoalkyl,mercaptoalkyl, hydroxyalkyl, mercaptoalkoxy, sulfonylalkyl,sulfonylaryl, alkylcarbonyl, and alkylcarbonylalkyl may be optionallysubstituted with one or more substituents.

Acids and bases useful in the methods herein are known in the art. Acidcatalysts are any acidic chemical, which can be inorganic (e.g.,hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic(e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid,ytterbium triflate) in nature. Acids are useful in either catalytic orstoichiometric amounts to facilitate chemical reactions. Bases are anybasic chemical, which can be inorganic (e.g., sodium bicarbonate,potassium hydroxide) or organic (e.g., triethylamine, pyridine) innature. Bases are useful in either catalytic or stoichiometric amountsto facilitate chemical reactions.

Alkylating agents are any reagent that is capable of effecting thealkylation of the functional group at issue (e.g., oxygen atom of analcohol, nitrogen atom of an amino group). Alkylating agents are knownin the art, including in the references cited herein, and include alkylhalides (e.g., methyl iodide, benzyl bromide or chloride), alkylsulfates (e.g., methyl sulfate), or other alkyl group-leaving groupcombinations known in the art. Leaving groups are any stable speciesthat can detach from a molecule during a reaction (e.g., eliminationreaction, substitution reaction) and are known in the art, including inthe references cited herein, and include halides (e.g., I-, Cl—, Br—,F—), hydroxy, alkoxy (e.g., —OMe, —O-t-Bu), acyloxy anions (e.g., —OAc,—OC(O)CF₃), sulfonates (e.g., mesyl, tosyl), acetamides (e.g.,—NHC(O)Me), carbamates (e.g., N(Me)C(O)Ot-Bu), phosphonates (e.g.,—OP(O)(OEt)₂), water or alcohols (protic conditions), and the like.

In certain embodiments, substituents on any group (such as, for example,alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl,cycloalkyl, heterocycloalkyl) can be at any atom of that group, whereinany group that can be substituted (such as, for example, alkyl, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl,heterocycloalkyl) can be optionally substituted with one or moresubstituents (which may be the same or different), each replacing ahydrogen atom. Examples of suitable substituents include, but are notlimited to alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaralkyl, aryl, heteroaryl, halogen, haloalkyl, cyano,nitro, alkoxy, aryloxy, hydroxyl, hydroxylalkyl, oxo (i.e., carbonyl),carboxyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl,alkylcarbonyloxy, aryloxycarbonyl, heteroaryloxy, heteroaryloxycarbonyl,thio, mercapto, mercaptoalkyl, arylsulfonyl, amino, aminoalkyl,dialkylamino, alkylcarbonylamino, alkylaminocarbonyl,alkoxycarbonylamino, alkylamino, arylamino, diarylamino, alkylcarbonyl,or arylamino-substituted aryl; arylalkylamino, aralkylaminocarbonyl,amido, alkylaminosulfonyl, arylaminosulfonyl, dialkylaminosulfonyl,alkylsulfonylamino, arylsulfonylamino, imino, carbamido, carbamyl,thioureido, thiocyanato, sulfoamido, sulfonylalkyl, sulfonylaryl,mercaptoalkoxy, N-hydroxyamidinyl, or N′-aryl, N″-hydroxyamidinyl.

Compounds of the invention can be made by means known in the art oforganic synthesis. Methods for optimizing reaction conditions, ifnecessary minimizing competing by-products, are known in the art.Reaction optimization and scale-up may advantageously utilize high-speedparallel synthesis equipment and computer-controlled microreactors(e.g., Design And Optimization in Organic Synthesis, 2^(nd) Edition,Carlson R, Ed, 2005; Elsevier Science Ltd.; Jahnisch, K et al, Angew.Chem. Int. Ed. Engl. 2004 43: 406; and references therein). Additionalreaction schemes and protocols may be determined by the skilled artesianby use of commercially available structure-searchable database software,for instance, SciFinder® (CAS division of the American Chemical Society)and CrossFire Beilstein® (Elsevier MDL), or by appropriate keywordsearching using an internet search engine such as Google® or keyworddatabases such as the US Patent and Trademark Office text database. Theinvention includes the intermediate compounds used in making thecompounds of the formulae herein as well as methods of making suchcompounds and intermediates, including without limitation those asspecifically described in the examples herein.

The compounds herein may also contain linkages (e.g., carbon-carbonbonds) wherein bond rotation is restricted about that particularlinkage, e.g. restriction resulting from the presence of a ring ordouble bond. Accordingly, all cis/trans and E/Z isomers are expresslyincluded in the present invention. The compounds herein may also berepresented in multiple tautomeric forms, in such instances, theinvention expressly includes all tautomeric forms of the compoundsdescribed herein, even though only a single tautomeric form may berepresented. All such isomeric forms of such compounds herein areexpressly included in the present invention. All crystal forms andpolymorphs of the compounds described herein are expressly included inthe present invention. Also embodied are extracts and fractionscomprising compounds of the invention. The term isomers is intended toinclude diastereoisomers, enantiomers, regioisomers, structural isomers,rotational isomers, tautomers, and the like. For compounds which containone or more stereogenic centers, e.g., chiral compounds, the methods ofthe invention may be carried out with an enantiomerically enrichedcompound, a racemate, or a mixture of diastereomers.

Preferred enantiomerically enriched compounds have an enantiomericexcess of 50% or more, more preferably the compound has an enantiomericexcess of 60%, 70%, 80%, 90%, 95%, 98%, or 99% or more. In preferredembodiments, only one enantiomer or diastereomer of a chiral compound ofthe invention is administered to cells or a subject.

Pharmaceutical Compositions

In one aspect, the invention provides a pharmaceutical compositioncomprising a compound of any formulae herein and a pharmaceuticallyacceptable carrier.

In another embodiment, the invention provides a pharmaceuticalcomposition further comprising an additional therapeutic agent. In afurther embodiment, the additional therapeutic agent is an anti-canceragent, antifungal agent, cardiovascular agent, antiinflammatory agent,chemotherapeutic agent, an anti-angiogenesis agent, cytotoxic agent, ananti-proliferation agent, metabolic disease agent, opthalmologic diseaseagent, central nervous system (CNS) disease agent, urologic diseaseagent, or gastrointestinal disease agent.

In one aspect, the invention provides a kit comprising an effectiveamount of a compound of any formulae herein, in unit dosage form,together with instructions for administering the compound to a subjectsuffering from or susceptible to a metalloenzyme-mediated disease ordisorder, including cancer, solid tumor, cardiovascular disease,inflammatory disease, infectious disease. In other embodiments thedisease, disorder or symptom thereof is metabolic disease, opthalmologicdisease, central nervous system (CNS) disease, urologic disease, orgastrointestinal disease.

The term “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable carrier” is meant to include salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds described herein.When compounds of the present invention contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydroiodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, e.g., Berge et al.,Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts. Other pharmaceutically acceptable carriersknown to those of skill in the art are suitable for the presentinvention.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

The invention also provides a pharmaceutical composition, comprising aneffective amount a compound described herein and a pharmaceuticallyacceptable carrier. In an embodiment, compound is administered to thesubject using a pharmaceutically-acceptable formulation, e.g., apharmaceutically-acceptable formulation that provides sustained deliveryof the compound to a subject for at least 12 hours, 24 hours, 36 hours,48 hours, one week, two weeks, three weeks, or four weeks after thepharmaceutically-acceptable formulation is administered to the subject.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of this invention may bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic(or unacceptably toxic) to the patient.

In use, at least one compound according to the present invention isadministered in a pharmaceutically effective amount to a subject in needthereof in a pharmaceutical carrier by intravenous, intramuscular,subcutaneous, or intracerebro ventricular injection or by oraladministration or topical application. In accordance with the presentinvention, a compound of the invention may be administered alone or inconjunction with a second, different therapeutic. By “in conjunctionwith” is meant together, substantially simultaneously or sequentially.In one embodiment, a compound of the invention is administered acutely.The compound of the invention may therefore be administered for a shortcourse of treatment, such as for about 1 day to about 1 week. In anotherembodiment, the compound of the invention may be administered over alonger period of time to ameliorate chronic disorders, such as, forexample, for about one week to several months depending upon thecondition to be treated.

By “pharmaceutically effective amount” as used herein is meant an amountof a compound of the invention, high enough to significantly positivelymodify the condition to be treated but low enough to avoid serious sideeffects (at a reasonable benefit/risk ratio), within the scope of soundmedical judgment. A pharmaceutically effective amount of a compound ofthe invention will vary with the particular goal to be achieved, the ageand physical condition of the patient being treated, the severity of theunderlying disease, the duration of treatment, the nature of concurrenttherapy and the specific compound employed. For example, atherapeutically effective amount of a compound of the inventionadministered to a child or a neonate will be reduced proportionately inaccordance with sound medical judgment. The effective amount of acompound of the invention will thus be the minimum amount which willprovide the desired effect.

A decided practical advantage of the present invention is that thecompound may be administered in a convenient manner such as byintravenous, intramuscular, subcutaneous, oral orintra-cerebroventricular injection routes or by topical application,such as in creams or gels. Depending on the route of administration, theactive ingredients which comprise a compound of the invention may berequired to be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions which mayinactivate the compound. In order to administer a compound of theinvention by other than parenteral administration, the compound can becoated by, or administered with, a material to prevent inactivation.

The compound may be administered parenterally or intraperitoneally.Dispersions can also be prepared, for example, in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils.

Some examples of substances which can serve as pharmaceutical carriersare sugars, such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethycellulose, ethylcellulose and cellulose acetates; powderedtragancanth; malt; gelatin; talc; stearic acids; magnesium stearate;calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil,sesame oil, olive oil, corn oil and oil of theobroma; polyols such aspropylene glycol, glycerine, sorbitol, manitol, and polyethylene glycol;agar; alginic acids; pyrogen-free water; isotonic saline; and phosphatebuffer solution; skim milk powder; as well as other non-toxic compatiblesubstances used in pharmaceutical formulations such as Vitamin C,estrogen and echinacea, for example. Wetting agents and lubricants suchas sodium lauryl sulfate, as well as coloring agents, flavoring agents,lubricants, excipients, tableting agents, stabilizers, anti-oxidants andpreservatives, can also be present. Solubilizing agents, including forexample, cremaphore and beta-cyclodextrins can also used in thepharmaceutical compositions herein.

Pharmaceutical compositions comprising the active compounds of thepresently disclosed subject matter (or prodrugs thereof) can bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making levigating, emulsifying, encapsulating, entrapping orlyophilization processes. The compositions can be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically.

Pharmaceutical compositions of the presently disclosed subject mattercan take a form suitable for virtually any mode of administration,including, for example, topical, ocular, oral, buccal, systemic, nasal,injection, transdermal, rectal, vaginal, and the like, or a formsuitable for administration by inhalation or insufflation.

For topical administration, the active compound(s) or prodrug(s) can beformulated as solutions, gels, ointments, creams, suspensions, and thelike.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal, oral, or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions also can contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection canbe presented in unit dosage form (e.g., in ampules or in multidosecontainers) and can contain added preservatives.

Alternatively, the injectable formulation can be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, and the like,before use. To this end, the active compound(s) can be dried by anyart-known technique, such as lyophilization, and reconstituted prior touse.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions can take theform of, for example, lozenges, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets can be coated by methods well known in theart with, for example, sugars or enteric coatings.

Liquid preparations for oral administration can take the form of, forexample, elixirs, solutions, syrups or suspensions, or they can bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl p-hydroxybenzoates or sorbic acid). The preparations also cancontain buffer salts, preservatives, flavoring, coloring and sweeteningagents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound or prodrug, as is well known.

For buccal administration, the compositions can take the form of tabletsor lozenges formulated in a conventional manner.

For rectal and vaginal routes of administration, the active compound(s)can be formulated as solutions (for retention enemas), suppositories, orointments containing conventional suppository bases, such as cocoabutter or other glycerides.

For nasal administration or administration by inhalation orinsufflation, the active compound(s) or prodrug(s) can be convenientlydelivered in the form of an aerosol spray from pressurized packs or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or othersuitable gas. In the case of a pressurized aerosol, the dosage unit canbe determined by providing a valve to deliver a metered amount. Capsulesand cartridges for use in an inhaler or insufflator (for examplecapsules and cartridges comprised of gelatin) can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

A specific example of an aqueous suspension formulation suitable fornasal administration using commercially-available nasal spray devicesincludes the following ingredients: active compound or prodrug (0.5-20mg/ml); benzalkonium chloride (0.1-0.2 mg/mL); polysorbate 80 (TWEEN®80; 0.5-5 mg/ml); carboxymethylcellulose sodium or microcrystallinecellulose (1-15 mg/ml); phenylethanol (1-4 mg/ml); and dextrose (20-50mg/ml). The pH of the final suspension can be adjusted to range fromabout pH5 to pH7, with a pH of about pH 5.5 being typical.

For prolonged delivery, the active compound(s) or prodrug(s) can beformulated as a depot preparation for administration by implantation orintramuscular injection. The active ingredient can be formulated withsuitable polymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, e.g., as a sparingly soluble salt. Alternatively,transdermal delivery systems manufactured as an adhesive disc or patchwhich slowly releases the active compound(s) for percutaneous absorptioncan be used. To this end, permeation enhancers can be used to facilitatetransdermal penetration of the active compound(s). Suitable transdermalpatches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat.No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S.Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189;U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S. Pat. No.5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475, each ofwhich is incorporated herein by reference in its entirety.

Alternatively, other pharmaceutical delivery systems can be employed.Liposomes and emulsions are well-known examples of delivery vehiclesthat can be used to deliver active compound(s) or prodrug(s). Certainorganic solvents such as dimethylsulfoxide (DMSO) also can be employed.

The pharmaceutical compositions can, if desired, be presented in a packor dispenser device which can contain one or more unit dosage formscontaining the active compound(s). The pack can, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration.

The active compound(s) or prodrug(s) of the presently disclosed subjectmatter, or compositions thereof, will generally be used in an amounteffective to achieve the intended result, for example in an amounteffective to treat or prevent the particular disease being treated. Thecompound(s) can be administered therapeutically to achieve therapeuticbenefit or prophylactically to achieve prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated and/or eradication or amelioration ofone or more of the symptoms associated with the underlying disorder suchthat the patient reports an improvement in feeling or condition,notwithstanding that the patient can still be afflicted with theunderlying disorder. For example, administration of a compound to apatient suffering from an allergy provides therapeutic benefit not onlywhen the underlying allergic response is eradicated or ameliorated, butalso when the patient reports a decrease in the severity or duration ofthe symptoms associated with the allergy following exposure to theallergen. As another example, therapeutic benefit in the context ofasthma includes an improvement in respiration following the onset of anasthmatic attack, or a reduction in the frequency or severity ofasthmatic episodes. Therapeutic benefit also includes halting or slowingthe progression of the disease, regardless of whether improvement isrealized.

For prophylactic administration, the compound can be administered to apatient at risk of developing one of the previously described diseases.A patient at risk of developing a disease can be a patient havingcharacteristics placing the patient in a designated group of at riskpatients, as defined by an appropriate medical professional or group. Apatient at risk may also be a patient that is commonly or routinely in asetting where development of the underlying disease that may be treatedby administration of a metalloenzyme inhibitor according to theinvention could occur. In other words, the at risk patient is one who iscommonly or routinely exposed to the disease or illness causingconditions or may be acutely exposed for a limited time. Alternatively,prophylactic administration can be applied to avoid the onset ofsymptoms in a patient diagnosed with the underlying disorder.

The amount of compound administered will depend upon a variety offactors, including, for example, the particular indication beingtreated, the mode of administration, whether the desired benefit isprophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, the bioavailability ofthe particular active compound, and the like. Determination of aneffective dosage is well within the capabilities of those skilled in theart.

Effective dosages can be estimated initially from in vitro assays. Forexample, an initial dosage for use in animals can be formulated toachieve a circulating blood or serum concentration of active compoundthat is at or above an IC50 of the particular compound as measured in asin vitro assay, such as the in vitro fungal MIC or MFC and other invitro assays described in the Examples section. Calculating dosages toachieve such circulating blood or serum concentrations taking intoaccount the bioavailability of the particular compound is well withinthe capabilities of skilled artisans. For guidance, see Fingl &Woodbury, “General Principles,” In: Goodman and Gilman's ThePharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46, latestedition, Pagamonon Press, and the references cited therein, which areincorporated herein by reference.

Initial dosages also can be estimated from in vivo data, such as animalmodels. Animal models useful for testing the efficacy of compounds totreat or prevent the various diseases described above are well-known inthe art.

Dosage amounts will typically be in the range of from about 0.0001 or0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but can be higher orlower, depending upon, among other factors, the activity of thecompound, its bioavailability, the mode of administration, and variousfactors discussed above. Dosage amount and interval can be adjustedindividually to provide plasma levels of the compound(s) which aresufficient to maintain therapeutic or prophylactic effect. In cases oflocal administration or selective uptake, such as local topicaladministration, the effective local concentration of active compound(s)cannot be related to plasma concentration. Skilled artisans will be ableto optimize effective local dosages without undue experimentation.

The compound(s) can be administered once per day, a few or several timesper day, or even multiple times per day, depending upon, among otherthings, the indication being treated and the judgment of the prescribingphysician.

Preferably, the compound(s) will provide therapeutic or prophylacticbenefit without causing substantial toxicity. Toxicity of thecompound(s) can be determined using standard pharmaceutical procedures.The dose ratio between toxic and therapeutic (or prophylactic) effect isthe therapeutic index. Compounds(s) that exhibit high therapeuticindices are preferred.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof. Therecitation of an embodiment herein includes that embodiment as anysingle embodiment or in combination with any other embodiments orportions thereof.

Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) in the manufacture of amedicament for use in the treatment of a metalloenzyme-mediated disorderor disease. Another object of the present invention is the use of acompound as described herein (e.g., of any formulae herein) for use inthe treatment of a metalloenzyme-mediated disorder or disease. Anotherobject of the present invention is the use of a compound as describedherein (e.g., of any formulae herein) in the manufacture of anagricultural composition for use in the treatment or prevention of ametalloenzyme-mediated disorder or disease in agricultural or agrariansettings.

Agricultural Applications

The compounds and compositions herein can be used in methods ofmodulating metalloenzyme activity in a microorganism on a plantcomprising contacting a compound (or composition) herein with the plant(e.g., seed, seedling, grass, weed, grain). The compounds andcompositions herein can be used to treat a plant, field or otheragricultural area (e.g., as herbicides, pesticides, growth regulators,etc.) by administering the compound or composition (e.g., contacting,applying, spraying, atomizing, dusting, etc.) to the subject plant,field or other agricultural area. The administration can be either pre-or post-emergence. The administration can be either as a treatment orpreventative regimen.

EXAMPLES

The present invention will now be demonstrated using specific examplesthat are not to be construed as limiting.

General Experimental Procedures

X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto-sample positioning and a HiStar2-dimensional area detector. X-ray optics consists of a single Göbelmultilayer mirror coupled with a pinhole collimator of 0.3 mm. A weeklyperformance check is carried out using a certified standard NIST 1976Corundum (flat plate). The beam divergence, i.e. the effective size ofthe X-ray beam on the sample, was approximately 4 mm. A θ-θ continuousscan mode was employed with a sample—detector distance of 20 cm whichgives an effective 20 range of 3.2°-29.7°. Typically the sample would beexposed to the X-ray beam for 120 seconds. The software used for datacollection was GADDS for XP/2000 4.1.43 and the data were analysed andpresented using Diffrac Plus EVA v13.0.0.2 or v15.0.0.0. Samples rununder ambient conditions were prepared as flat plate specimens usingpowder as received without grinding. Approximately 1-2 mg of the samplewas lightly pressed on a glass slide to obtain a flat surface. Samplesrun under non-ambient conditions were mounted on a silicon wafer withheat-conducting compound. The sample was then heated to the appropriatetemperature at 30° C./min and subsequently held isothermally for 1minute before data collection was initiated.

X-Ray Powder Diffraction patterns were also collected on a Bruker D8diffractometer using Cu Kα radiation (40 kV, 40 mA), 0-20 goniometer,and divergence of V4 and receiving slits, a Ge monochromator and aLynxeye detector. The instrument is performance checked using acertified Corundum standard (NIST 1976). The software used for datacollection was Diffrac Plus XRD Commander v2.6.1 and the data wereanalysed and presented using Diffrac Plus EVA v13.0.0.2 or v15.0.0.0.Samples were run under ambient conditions as flat plate specimens usingpowder as received. The sample was gently packed into a cavity cut intopolished, zero-background (510) silicon wafer. The sample was rotated inits own plane during analysis. The details of the data collection are:Angular range: 2 to 42° 2θ; Step size: 0.05° 2θ; Collection time: 0.5s/step.

Single crystal X-ray diffraction data (SCXRD) were collected on a OxfordDiffraction Supernova Dual Source, Cu at Zero, Atlas CCD diffractometerequipped with an Oxford Cryosystems Cobra cooling device. The data wascollected using CuKα radiation. Structures were typically solved usingeither the SHELXS or SHELXD programs and refined with the SHELXL programas part of the Bruker AXS SHELXTL suite (V6.10). Unless otherwisestated, hydrogen atoms attached to carbon were placed geometrically andallowed to refine with a riding isotropic displacement parameter.Hydrogen atoms attached to a heteroatom were located in a differenceFourier synthesis and were allowed to refine freely with an isotropicdisplacement parameter.

Differential Scanning Calorimetry (Modulated temperature DSC) wascarried out using an underlying heating rate of 2° C./min andtemperature modulation parameters of ±0.636° C. or ±1.272° C.(amplitude) every 60 seconds (period). The instrument control softwarewas Advantage for Q Series v2.8.0.394 and Thermal Advantage v5.2.6 andthe data were analysed using Universal Analysis v4.7A or v4.4A.

DSC data was also collected on a Mettler DSC 823E equipped with a 34position auto-sampler. The instrument was calibrated for energy andtemperature using certified indium. Typically 0.5-3 mg of each sample,in a pin-holed aluminium pan, was heated at 10° C./min from 25° C. to350° C. A nitrogen purge at 50 ml/min was maintained over the sample.The instrument control and data analysis software was STARe v9.20.

NMR spectra were collected on a Bruker 400 MHz instrument equipped withan auto-sampler and controlled by a DRX400 console. Automatedexperiments were acquired using ICON-NMR v4.0.7 running with Topspinv1.3 using the standard Bruker loaded experiments. For non-routinespectroscopy, data were acquired through the use of Topspin alone.Samples were prepared in deuterated chloroform (CDCl₃), unless otherwisestated. Off-line analysis was carried out using Topspin v1.3 or ACDSpecManager v12.5.

Fourier Transform—Infra-red (FTIR) Data were collected on a Perkin-ElmerSpectrum One fitted with a universal Attenuated Total Reflectance (ATR)sampling accessory. The data were collected and analysed using Spectrumv10.0.1 software.

Thermo-gravimetric analysis (TGA) data were collected on a MettlerTGA/SDTA 851e equipped with a 34 position auto-sampler. The instrumentwas temperature calibrated using certified indium. Typically 2-8 mg ofeach sample was loaded onto a pre-weighed aluminium crucible and washeated at 10° C./min from ambient temperature to 350° C. A nitrogenpurge at 50 ml/min was maintained over the sample. The instrumentcontrol and data analysis software was STARe v9.20.

The water content of each sample was measured by Karl Fischer titration(KF) on a Metrohm 874 Oven Sample Processor at 150° C. with 851 TitranoCoulometer using Hydranal Coulomat AG oven reagent and nitrogen purge.Weighed solid samples were introduced into a sealed sample vial.Approximately 10 mg of sample was used per titration and duplicatedeterminations were made.

Gravimetric Vapor Sorption (GVS) isotherms were obtained using a SMS DVSIntrinsic moisture sorption analyser, controlled by DVS IntrinsicControl software v1.0.1.2 (or v 1.0.1.3). The sample temperature wasmaintained at 25° C. by the instrument controls. The humidity wascontrolled by mixing streams of dry and wet nitrogen, with a total flowrate of 200 ml/min The relative humidity was measured by a calibratedRotronic probe (dynamic range of 1.0-100% RH), located near the sample.The weight change, (mass relaxation) of the sample as a function of % RHwas constantly monitored by the microbalance (accuracy±0.005 mg).Typically 5-20 mg of sample was placed in a tared mesh stainless steelbasket under ambient conditions. The sample was loaded and unloaded at40% RH and 25° C. (typical room conditions). A moisture sorptionisotherm was performed as outlined below (2 scans giving 1 completecycle). The standard isotherm was performed at 25° C. at 10% RHintervals over a 0-90% RH range. Data analysis was undertaken inMicrosoft Excel using DVS Analysis Suite v6.2 (or 6.1 or 6.0).

Method Parameters for SMS DVS Intrinsic Experiments Parameters ValuesAdsorption - Scan 1 40-90 Desorption/Adsorption - Scan 2 90-0, 0-40Intervals (% RH) 10 Number of Scans 4 Flow rate (ml/min) 200 Temperature(° C.) 25 Stability (° C./min) 0.2 Sorption Time (hours) 6 hour time outThe sample was recovered after completion of the isotherm andre-analysed by XRPD.

Definitions of variables in the structures in schemes herein arecommensurate with those of corresponding positions in the formulaedelineated herein.

Synthesis of 1 or 1a

Example 1

Preparation of Compound 1 X-Hydrate

Compound 1 and its preparation are described in the art, including inU.S. Pat. No. 8,236,962 (incorporated by reference herein). Compound 1can then be partitioned between ethanol and water to afford Compound 1X-hydrate.

Example 2

Compound 1 Anhydrous Form Recrystallization

Compound 1 X-hydrate (29.1 g, 28.0 g contained 1) was suspended in2-propanol (150 ml) and heated to 56° C. The solution was filteredthrough a 0.45 μm Nylon membrane with 2-propanol rinses. The combinedfiltrate was concentrated to 96.5 g of a light amber solution. Thesolution was transferred to a 1-L flask equipped with overhead stirring,thermocouple and addition funnel, using 2-propanol (30 ml total) tocomplete the transfer. The combined solution contained about 116 ml2-propanol.

The solution was heated to 50° C. and n-heptane (234 ml) was added over22 minutes. The resulting hazy mixture was seeded with 1 anhydrous form.After about 1 hour a good suspension had formed. Additional n-heptane(230 ml) was added over 48 minutes. Some granular material separated butmost of the suspension was a finely divided pale beige solid. Afterabout ½ hour at 50° C. the suspension was cooled at 10° C./h to roomtemperature and stirred overnight. The product was collected at 22° C.on a vacuum filter and washed with 1:4 (v/v) 2-PrOH/n-heptane (2×50 ml).After drying on the filter for 1-2 hours the weight of product was 25.5g. The material was homogenized in a stainless steel blender topulverize and blend the more granular solid component. The resultingpale beige powder (25.37 g) was dried in a vacuum oven at 50° C. The dryweight was 25.34 g. The residual 2-propanol and n-heptane were estimatedat <0.05 wt % each by ¹H NMR analysis. The yield was 90.5% aftercorrecting the X-hydrate for solvent and water content. Residual Pd was21 ppm. The water content was 209 ppm by KF titration. The melting pointwas 100.7° C. by DSC analysis.

TABLE 1 Data for the isolated and dried Compound 1 - X-hydrate andanhydrous forms Yield M.P. Pd Purity Chiral H₂O Residual Solvent Form(%) (° C.) (ppm) (A %) (A %) (wt %) (wt %) X-Hydrate 91.0 65 48 99.4 NA2.16% Ethanol - 1.7 wt % Anhydrous 90.5 101 21 99.8 >99.9 209 ppm2-PrOH, heptane <0.1 wt % M.P. by DSC; Pd by ICP; Purity by the API HPLCmethod; Chiral purity by HPLC; water content by KF titration; residualsolvent estimated from ¹H NMR.

TABLE 2 Characterisation Data for Compounds 1 (X-hydrate) and 1(anhydrous) Technique X-hydrate 1 Anhydrous 1 XRPD Crystalline(Pattern 1) Crystalline (anhydrous) XRPD stability 40° C. & 75% RH Nochange in crystalline form No change in crystalline form post 2 weeks at25° C. & 97% RH No change in crystalline form No change in crystallineform ^(1H)NMR Consistent with 1 structure Consistent with 1 structure(0.2 mol of EtOH) (0.02 mol of heptane) Thermal DSC 62° C. (ΔH = −99J/g) Melt onset ~99° C. (ΔH = −37 J/g) TGA 4.2% (40° C.-130° C.)Decomposition onset at ~190° C. Decomposition onset at ~190° C. HPLC99.88% by area 99.89% by area PLM Needle like crystals Needle likecrystals and agglomerates particle size >100 μm particle size range from5 μm-100 μm GVS 0.59% w/w water uptake at 90% RH. 0.14% w/w water uptakeat 90% RH. No sample hysteresis No sample hysteresis XRPD No form changeafter GVS experiment No form change after GVS experiment post GVS KF2.4% w/w H₂0 Not obtained Solubility <0.001 mg/ml <0.001 mg/ml pH ofsaturated solution = 8.6 pH of saturated solution = 8.7 FT-IR SpectralPattern 1 Spectral Pattern 2 Charcteristic bands/cm⁻¹: Charcteristicbands/cm⁻¹: 3499, 3378, 3213, 3172 3162 1612, 1598, 1588, 1522, 15021610, 1518, 1501 931, 903, 875, 855, 828, 816 927, 858, 841, 829, 812

TABLE 3 Single Crystal Structure of 1 Anhydrous Form Molecular formulaC₂₃H₁₆F₇N₅O₂ Molecular weight 527.41 Crystal system Monoclinic Spacegroup P2(1) a 10.9345(6) Å, α 90°, b 8.3118(5) Å, β 97.286(5)°, c24.5678(14) Å, γ 90° V 2214.8(2) Å³ Z 4 D_(c) 1.582 g · cm⁻³ μ 1.271mm⁻¹ Source, λ Cu—Kα, 1.54178 Å F(000) 1072 T 100(2) K CrystalColourless plate, 0.3 × 0.2 × 0.08 mm Data truncated to 0.80 Å θ_(max)74.49° Completeness 99.5% Reflections 23372 Unique reflections 8807R_(int) 0.0327

The structure solution of Compound 1 anhydrous form was obtained bydirect methods, full-matrix least-squares refinement on F² withweighting w⁻¹=σ²(F_(o) ²)+(0.0474 P)²+(0.3258 P), where P=(F_(o) ²+2F_(c) ²)/3, anisotropic displacement parameters, empirical absorptioncorrection using spherical harmonics, implemented in SCALE3 ABSPACKscaling algorithm. Final wR²={Σ[w(F_(o) ²−F_(c) ²)²]/Σ[w(F_(o)²)²]^(1/2)}=0.0877 for all data, conventional R₁=0.0343 on F values of8390 reflections with F_(o)>4a(F_(o)), S=1.051 for all data and 675parameters. Final Δ/a (max) 0.001, Δ/a(mean), 0.000. Final differencemap between +0.311 and −0.344 e Å⁻³.

Below shows a view of two molecules of Compound 1 in the asymmetric unitof the anhydrous form showing the numbering scheme employed (FIG. 9).Anisotropic atomic displacement ellipsoids for the non-hydrogen atomsare shown at the 50% probability level. Hydrogen atoms are displayedwith an arbitrarily small radius. The absolute configuration of themolecules has been determined to be R.

Example 3

Compound 1 Ethanol Solvate Recrystallization

Compound 1 X-hydrate (50 mg) was suspended in ˜40 volumes of 15%H₂O/EtOH. The suspension was then placed in an incubation chamber formaturation. The maturation protocol involved treating the suspension toa two-temperature cycle of 50° C./ambient temperature at 8 hours percycle for 3 days with constant agitation. After maturation, thesuspension was cooled in a fridge at 4° C. for up to 2 days to encouragethe formation of crystals. Then, the solvent was removed at RT and thesample was vacuum dried at 30° C.-35° C. for up to 1 day. Suitablecrystals formed on cooling were harvested and characterized.

TABLE 4 Single Crystal Structure of 1 Ethanol solvate Molecular formulaC₂₅H₂₂F₇N₅O₃ Molecular weight 573.48 Crystal system Orthorhombic Spacegroup P2(1)2(1)2(1) a 7.4595(3) Å, α 90°, b 14.4651(6) Å, β 90°, c22.6496(8) Å, γ 90° V 2443.95(17) Å³ Z 4 D_(c) 1.559 g · cm⁻³ μ 1.232mm⁻¹ Source, λ Cu—Kα 1.54178 Å F(000) 1176 T 100(2) K Crystal Colourlesscolumnar, 0.25 × 0.05 × 0.05 mm Data truncated to 0.80 Å θ_(max) 74.44°Completeness 99.3% Reflections 12874 Unique reflections 4872 R_(int)0.0264

The structure solution of Compound 1 ethanol solvate was obtained bydirect methods, full-matrix least-squares refinement on F² withweighting w⁻¹=σ²(F_(o) ²)+(0.0450 P)²+(0.5000 P), where P=(F_(o) ²+2F_(c) ²)/3, anisotropic displacement parameters, empirical absorptioncorrection using spherical harmonics, implemented in SCALE3 ABSPACKscaling algorithm. Final wR²={Σ[w(F_(o) ²−F_(c) ²)²]/Σ[w(F_(o)²)²]^(1/2)}=0.0777 for all data, conventional R₁=0.0272 on F values of4591 reflections with F_(o)>4σ(F_(o)), S=1.006 for all data and 370parameters. Final Δ/σ (max) 0.000, Δ/σ(mean), 0.000. Final differencemap between +0.217 and −0.199 e Å⁻³.

Below shows a view of the asymmetric unit of the ethanol solvate fromthe crystal structure showing the numbering scheme employed (FIG. 10).Anisotropic atomic displacement ellipsoids for the non-hydrogen atomsare shown at the 50% probability level. Hydrogen atoms are displayedwith an arbitrarily small radius. The asymmetric unit showsstoichiometry of 1:1 for solvent of crystallisation to Compound 1.

Example 4

Compound 1 1.5 Hydrate Recrystallization

Compound 1 X-hydrate (50 mg) was suspended in ˜40 volumes of 15%H₂O/IPA. The suspension was then placed in an incubation chamber formaturation. The maturation protocol involved treating the suspension toa two-temperature cycle of 50° C./ambient temperature at 8 hours percycle for 3 days with constant agitation. After maturation, thesuspension was cooled in a fridge at 4° C. for up to 2 days to encouragethe formation of crystals. Then, the solvent was removed at RT and thesample was vacuum dried at 30° C.-35° C. for up to 1 day. Suitablecrystals formed on cooling were harvested and characterized.

TABLE 5 Single Crystal Structure of 1 1.5 Hydrate Molecular formulaC₂₃H₁₈F₇N₅O_(3.50) Molecular weight 553.42 Crystal system MonoclinicSpace group C2 a 22.409(3) Å, α 90°, b 7.5646(8) Å, β 103.372(15)°, c13.979(2) Å, γ 90° V 2305.4(5) Å³ Z 4 D_(c) 1.595 g · cm⁻³ μ 1.3 mm⁻¹Source, λ Cu—Kα 1.54178 Å F(000) 1128 T 100(2) K Crystal Colourlesscolumnar, 0.7 × 0.5 × 0.3 mm Data truncated to 0.80 Å θ_(max) 58.93°Completeness 98.6% Reflections 3736 Unique reflections 2214 R_(int)0.0454

The structure solution of Compound 1 1.5 hydrate was obtained by directmethods, full-matrix least-squares refinement on F² with weightingw⁻¹=σ²(F_(o) ²)+(0.1269 P)²+(0.0000 P), where P=(F_(o) ²+2 F_(c) ²)/3,anisotropic displacement parameters, empirical absorption correctionusing spherical harmonics, implemented in SCALE3 ABSPACK scalingalgorithm. Final wR²={Σ[w(F_(o) ²−F_(c) ²)²]/Σ[w(F_(o)²)²]^(1/2)}=0.1574 for all data, conventional R₁=0.0668 on F values of2106 reflections with F_(o)>4σ(F_(o)), S=1.106 for all data and 361parameters. Final Δ/σ (max) 0.000, Δ/σ(mean), 0.000. Final differencemap between +0.439 and −0.598 e Å⁻³. Below shows a view of theasymmetric unit of the 1.5 hydrate from the crystal structure showingthe numbering scheme employed (FIG. 11). Anisotropic atomic displacementellipsoids for the non-hydrogen atoms are shown at the 50% probabilitylevel. Hydrogen atoms are displayed with an arbitrarily small radius.The asymmetric unit shows stoichiometry of 1.5:1 for water to Compound1.

Example 5

Human Pharmacokinetic Comparison of Compound 1 X-Hydrate and Compound 1Anhydrous Form

Table 6 compares human multiple-dose pharmacokinetic (PK) parametersbetween dosing with Compound 1 X-hydrate and Compound 1 Anhydrous form.Compound 1 X-hydrate was dosed at 600 mg twice daily (bid) for threedays followed by dosing at 300 mg once daily (qd) for 10 days. Compound1 Anhydrous form was dosed at 300 mg qd for 14 days. Despite the higherinitial dosing amount and frequency (i.e., 600 mg bid) of Compound 1X-hydrate, Compound 1 Anhydrous form surprisingly displayed highermaximal concentration (C_(max)) and higher area-under-the-curve (AUC)than Compound 1 X-hydrate.

TABLE 6 Comparison of Multiple Dose PK between Compound 1 X-Hydrate andCompound 1 Anhydrous Polymorph C_(max) T_(max) AUC_(0-8 h) Test ArticleDose Regimen (ng/mL) (h) (ngh/mL) Compound 1 600 mg bid loading dose for2520 5 17600 X-Hydrate 3 days, then 300 mg qd maintenance for 10 daysCompound 1 300 mg qd for 14 days 4693 5 35653 AnhydrousFurther characterization of the various polymorph forms of compound 1are detailed in the accompanying figures.

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

The invention claimed is:
 1. A polymorph of a compound of formula 1,

characterized by at least one of: (i) a powdered X-ray diffractionpattern having two or more peaks expressed in degrees 2-theta±0.2° andselected from peaks delineated in the pattern of FIG. 1; or (ii) a DSCthermogram showing an endotherm at about 101-108° C.
 2. The polymorphcompound of claim 1, characterized by a powdered X-ray diffractionpattern having peaks expressed in degrees 2-theta±0.2° at each of about8.4, 11.2, 17.8, 19.1, and 22.3.
 3. The polymorph compound of claim 1,characterized by a melting point of about 101-108° C.
 4. The polymorphcompound of claim 1, having less than 0.5% water therein.
 5. Thepolymorph compound of claim 1, wherein the compound is essentiallysolvent free.
 6. The polymorph compound of claim 1, wherein the compoundhas less than 0.5 wt. % solvent, less than 0.25 wt. % solvent, or lessthan 0.10 wt. % solvent.